1.1 Field of the Invention
The present invention relates to the field of molecular biology. More specifically, it concerns methods for the incorporation of foreign DNA into the genome of monocotyledonous plants, and in particular, wheat. Provided herein are reproducible systems for genetically transforming wheat, methods of selecting stable genetic transformants from suspensions of transformed cells, and methods of producing fertile plants from the transformed cells. Exemplary methods include the use of Agrobacterium-mediated transformation to introduce nucleic acids into cells, and selectable and/or screenable marker systems, for example, genes which confer resistance (e.g., antibiotic, herbicide, etc.), or which contain an otherwise phenotypically observable trait. In other aspects, the invention relates to the production of stably transformed and fertile wheat plants, gametes and offspring from these plants.
1.2 Description of the Related Art
The entire text of U.S. patent application Ser. No. 08/329,742 filed Oct. 26, 1994 is hereby incorporated by reference in its entirety. During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, diseases and herbicides, and to modify biosynthetic processes to change the quality of plant products (Knutson et al., 1992; Piorer et al., 1992; Vasil et al., 1992). However, the availability of an efficient transformation method to introduce foreign DNA has been a substantial barrier for most monocot species, including maize, rice, oat, barley, and particularly wheat.
1.2.1 Available Methods for Transforming Monocotyledonous Plants
There have been many methods attempted for the transformation of monocotyledonous plants but only a few methods have resulted in stable transformation. Two methods are currently employed for most transgenic studies in monocot species: direct DNA transfer into isolated protoplasts and microprojectile-mediated DNA delivery (Shimamoto et al., 1989; Fromm et al., 1990). More recently, additional methods have also been developed for use in monocots. Following is a brief description of the methods that have resulted in stably transformed and fertile monocots capable of transferring genes to their progeny in a Mendelian fashion.
1.2.1.1 Biolistics
“Biolistics” is most widely used transformation method for monocotyledons. In the “biolistics” method microprojectile particles are coated with DNA and accelerated by a mechanical device to a speed high enough to penetrate the plant cell wall and nucleus (Intl. Pat. Appl. Publ. No. WO 91/02071). The foreign DNA gets incorporated into the host DNA and results in a transformed cell. There are many variations on the “biolistics” method (Sanford, 1990; Fromm et al., 1990; Christou et al., 1988; Sautter et al., 1991). This method has been successfully used to produce stably transformed monocotyledonous plants including rice, maize, wheat, barley, and oats (Christou et al., 1991; Gordon-Kamm et al., 1990; Vasil et al., 1992, 1993; Wan et al., 1993; Sommers et al., 1992).
The microprojectile-mediated DNA delivery method may use immature embryos or immature embryo derived calli as target tissues. Transgenic plants have been recovered from the microprojectile bombardment method in maize, oat, barley and wheat (Gordon-Kamm et al., 1990; Somers et al., 1992; Wan et al., 1994; Vasil et al., 1992).
The microprojectile bombardment method generally takes 10 to 15 months to obtain transgenic plants (Gordon-Kamm et al., 1990; Vasil et al., 1992). Even with the more recent improvements in transformation methods using immature embryos as target tissues, it still requires 4 to 6 months to recover transgenic plants (Weeks et al., 1993; Vasil et al., 1992; 1993; Becker et al., 1994). The transformation frequency by these methods is variable ranging from about one event from 100 to 1000 bombarded embryos.
1.2.1.2 Electroporation
The protoplast methods have been widely used in rice, where DNA is delivered to the protoplasts through liposomes, PEG, and electroporation. While a large number of transgenic plants have been recovered in several laboratories (Shimamoto et al., 1989; Datta et al., 1990), the protoplast methods require the establishment of long-term embryogenic suspension cultures. Some regenerants from protoplasts are infertile and phenotypically abnormal due to the long-term suspension culture (Davey et al., 1991; Rhodes et al., 1988). These procedures have been especially useful for rice and some grasses.
Transformation by electroporation involves the application of short, high voltage electric fields to create “pores” in the cell membrane through which DNA is taken-up. This method has been used to produce stably transformed monocotyledon plants, (Pasazkowski et al., 1985; Shillito et al., 1985; Fromm et al., 1986) especially from rice (Shimamoto et al., 1992; Datta et al., 1990, 1992; Hayakawa et al., 1992).
1.2.1.3 Chemical Treatment of Protoplasts
The polyethylene glycol (PEG) method is simply a chemical treatment in the presence of the protoplasts and the DNA (Shillito et al., 1985; Rhodes et al., 1988). The PEG facilitate the uptake of the DNA.
1.2.1.4 Other Methods
A number of other methods have been reported for the transformation of monocotyledon plants. The methods reported to produce fertile transgenic monocotyledon plants include the “pollen tube method” (Intl. Pat. Appl. Publ. No. WO 93/18168; Zahir, 1993, Luo and Wu, 1988) and macro-injection of DNA into floral tillers (Du et al., 1989; Picard et al., 1988; De la Pena et al., 1987) and tissue incubation of seeds in DNA solutions (Tofer et al., 1989). Direct injection of exogenous DNA into the endosperm of a fertilized plant ovule at the onset of embryogenesis was disclosed in Intl. Pat. Appl. Publ. No. WO 94/00583. Besides the protoplast and the biolistics methods of transformation other methods are not reproducible or predictable. There is usually evidence of expression but seldom is the DNA transmitted to the progeny.
1.3 Deficiencies in the Prior Art
The one important area where there has been little significant progress in the art has been the adaptation of bacterial-mediated methods of transformation in monocots. While widely useful in dicotyledonous plants, Agrobacterium-mediated gene transfer has been disappointing when adapted to use in monocots. There are several reports in the literature claiming Agrobacterium transformation of monocotyledons which are discussed in Intl. Pat. Appl. Publ. No. WO 94/0077. These are specifically the methods of Gould et al., 1991; Mooney et al., 1991; and Raineri et al., 1990, which claim Agrobacterium transformation of maize, rice and wheat. There is some evidence of gene transfer in these methods but they lack convincing evidence for transfer efficiency, reproducibility, and confirmation of gene transfer (Potrykus, 1990), and lack of transfer to the progeny when plants are produced. In the work of Gould where evidence of transformed plants was presented there was no Mendelian inheritance of the genes.
De LaFonteyne et al. (Intl. Pat. Appl. Publ. No. WO 92/06205) described a process for the transformation of maize cells using A. tumefaciens strains in combination with a transposon-mediated integration method, but the success of such methods in other species was not demonstrated.
Mooney et al. (1991) produced transformed cells from wheat embryos co-cultivated with A. tumefaciens but the frequency of transformation was very low and often unreproducible. Chan et al. (1993) subsequently attempted to produce transgenic rice plants using A. tumefaciens, but their methods have not been widely accepted owing to a lack of sufficient molecular and genetic evidence of transgenic plant production.
More recent attempts by Hiei et al, (1994) suggested that transgenic rice plants could be obtained following A. tumefaciens transformation, but that the particular bacterial strains used and the choice of bacterial vectors were critical for successfully obtaining transgenes. A recent paper by Ishida et al. (1996) indicated a high-efficiency transformation of maize was possible by co-culture of immature embryos with A. tumefaciens. In both reports on rice and the maize transformation, a super binary vector pTOK233 containing the virb, virC and virG genes was used to achieve high-efficiency transformation. A recent report by Saito et al. (Intl. Pat. Appl. Publ. No. WO 95/06722) discloses a method of transforming monocotyledons using scutellum of non-dedifferentiated immature embryos using A. tumefaciens. 
Despite the fact that wheat is the most widely-grown cereal crop in the world, unfortunately no convincing reports exist on the use of Agrobacterium transformation methods in the preparation of stable, fertile wheat transgenic plants. Likewise, no methods have been developed using immature embryonic or callus tissues for stable, high-frequency transformation of wheat. Therefore, what is lacking in the prior art is an Agrobacterium-mediated method for preparing fertile, transgenic wheat plants.